Endotoxins by definition are lipopolysaccharides or "LPS" constituents of the outer membrane of gram-negative bacteria. All endotoxins comprise a polysaccharide portion and a covalently bound lipid component called "lipid A"; and hence, they are chemically lipopolysaccharides. In common practice and in clinical practice, the terms "endotoxin" and "lipopolysaccharide" are often used interchangeably and synonymously; but in its strict denotative meaning, an endotoxin represents a complexed association of LPS with other outer membrane biochemical compositions and structures [Rietschel et al., Scan. J. Infect. Dis. (Suppl.) 31:8-21 (1982); Westphal et al., Advances In Immunopharmacology, Pergammon Press, 1985; Van DeVenter et al., Gastroenterology 94:825-831 (1988); and Reitschel et al., Infectious Disease Clinics Of North America 5:753-779 (1991)].
Endotoxins were first discovered and identified at the end of the 19th century when it was found that lysates of heat-inactivated Vibrio cholerae contained a toxic component which was capable of inducing shock and death in experimental animals. This heat-stable toxic was termed "endotoxin" to distinguish it from the heat-labile "exotoxins" which were actively secreted by the live bacteria. About the same time, other scientists independently isolated the same toxin and made two important contributions: First, it was observed that this endotoxin could be isolated from lysates of many different gram-negative bacteria, but never from similar preparations of gram-positive bacteria. Second, the remarkable pyrogenic properties of endotoxins were demonstrated. However, it was only in 1935 that research investigators, using a method of trichloracetic extraction, determined that the endotoxic activity of gram-negative bacterial lysates resides in an outer-membrane macromolecular complex of protein, lipid and polysaccharide. Twenty years later, the now classic studies and biochemical investigations of endotoxin were performed--which demonstrated that protein-free lipopolysaccharide possessed all the properties of the earlier prepared, crude endotoxin.
A detailed knowledge of the biochemistry of the cell wall of gram-negative bacteria is helpful in understanding the structural basis of the toxicity and also of the rationale for the conventionally known approaches to treatment of endotoxic shock. The cell wall of gram-negative bacteria is a complex structure composed of the innermost cytoplasmic membrane, the periplasm, the peptidoglycan layer, the outer membrane, and, in many instances, additional structures such as capsules, extracellular polysaccharide, fimbriae and flagella [Bayston, K. F. and J. Cohen, J. Med. Microbiol. 31:73-83 (1990)]. Endotoxin (LPS) is found exclusively in the outer membrane; and, specifically, only in the outer leaflet of this membrane. Here, LPS forms a hydrophobic barrier which restricts the entry of noxious substances such as bile salts, digestive enzymes and certain antibiotics; and enables the living bacterium to evade many innate host-defense factors including complement, lysozyme and cationic proteins. Endotoxin may also be found in a cell-free form which occurs after bacterial autolysis; as a result of exposure to cell-membrane toxins or antibiotics; during rapid (log-phase) growth; or when essential nutrients are depleted from the environment. All of these conditions typically arise during septicaemia [Brogden, K. A. and M. Phillips, Electron. Microsc. Rev. 1:261-277 (1988); Flynn et al., Infect. Immun. 56:2760-2762 (1988); and Shenep et al., J. Infect. Dis. 157:565-568 (1988)].
Endotoxins have been implicated in the pathogenesis of a variety of different clinical disorders. A representative, but incomplete listing of abnormal clinical conditions and specific disease states is given by Table A below.
TABLE A ______________________________________ Disease Association Reference ______________________________________ Septic shock, sepsis, infections. Gorelick et al., Infect Dis. Clin. N. Amer. 5:899-913 (1991). Various forms of liver disease; Nolan, J. P., Immunological e.g. - viral hepatitis, alcoholic liver Investigations 18:325-337 injury, liver failure, cirrhosis, (1989). Nolan, J. P. Hepatol- obstructive jaundice, toxic livery ogy 10:887-891 (1989); Pappo injury; total parenteral nutrition et al., J. Parent. Enteral. associated. Nutr. 16:529-532 (1992). Bowel disease; e.g. - Crohn's Aoki, K., Acta Med disease, ulcerative colitis, ischemia, Okayama 32:207-216 (1978). bowel obstruction, ilueus. Papa et al., J Surg Res 35: 264-269 (1983). Rosher et al., Am J Surg 155:348-355 (1988). Kidney disease; e.g. - acute renal Wardle, E. N., Quart. J. failure, glomerulonephritis. Med. 44:389-398 (1975). Tomosugi et al., Immuno- biology 175:104 (1987). Lung disease; e.g. - adult respira- Brigham, K. L., Am. Rev. tory distress syndrome, pneumonia. Resp. Dis. 133:913-927 (1986). Trauma; e.g. - abdominal/chest Kelley, et al., Surg. trauma, surgery, colonoscopy. Gynecol-Obstet. 161:332-334 (1985). Radiation injury. Maxwell, et al., Lancet 1:1148-1149 (1986). Graft versus host disease. Moore, et al., Transplan- tation 43:731-736 (1987). Toxic-shock syndrome. Stone, et al., J. Infect. Dis. 155:682-689 (1987). Immunocompromised patients with Harris, et al., J. Clin. Pathol. fever. 37:467-70 (1984). Neonatal necrotizing enterocolitis. Schiefe, et al., Am. J. Clin. Pathol. 83:227-229 (1985). Acute pancreatis Exley, et al., Gut. 33:116-1128 (1992). Liver transplantation Yokoyama, et al., Transpl. Proc. 21:3833-3841 (1989); Miyata, et al., Lancet 2:189-191 (1989). Heat stroke Anon, Lancet 2:1137-1138 (1989). ______________________________________
Of the representative abnormal clinical conditions and disease states mediated by endotoxins, gram-negative septic shock is probably the most familiar to physicians and is the setting in which the role of endotoxin is most clearly established by empirical evidence [Morrison, D. C. and J. L. Ryan, Ann. Rev. Med. 38:417-432 (1987)]. Septic shock is estimated to occur in about 20-40% of patients with gram-negative septicaemia. Of these, about 75% die despite the use of potent antibiotics and intensive-care facilities--the rationale for this phenomenon being that while antibiotics are very effective at killing bacteria, these compositions have no activity against endotoxins or the host-derived factors which are now thought to mediate the toxic effects of endotoxin [Bayston, K. F. and J. Cohen, J. Med. Microbiol. 31:73-83 (1990) and the references cited therein].
Another major area of research investigations regarding the physiological and pharmacological effects of endotoxins is alcoholic liver disease. The role of endotoxin in alcoholic liver disease, hepatic failure, and hepatic necrosis secondary to toxic and infectious agents has been recognized for many years. Representative of these research investigations are the following publications: Formal et al., Proc. Soc. Exp. Biol. Med. 103:451-418 (1960); Galanos et al., Proc. Natl. Acad. Sci. U.S.A. 76:939-943 (1979); Camara et al., Proc. Soc. Exp. Biol. Med. 172:255-259 (1983); Nolan J. P. and D. S. Camaria, Immun. Invest. 18:325-337 (1989); Shibayana et al., Exp. Mol. Pathol. 55:196-202 (1991); Rutenburg et al., J. Exp. Med. 106:1-13 (1957); Broitman et al., J. Exp. Med. 119:633-641 (1964); Nolan, J. P. and N. V. Ali, Proc. Soc. Exp. Biol. Med. 129:29-31 (1968); Bhagwandeen et al., J. Pathol. 151:47-53 (1987); Arai et al., Hepatology 9:846- 851 (1989); Nolan, J. P., Hepatology 10:887-891 (1989); Van Leeuwen et al., Surgery 110:169-175 (1991); Nanji et al., AASLD A859 (April 1992).
One of the curiosities regarding endotoxins clinically and physiologically are those areas and organs in which endotoxin is typically present or explicitly absent in the normal living individual--in comparison to those tissues and organs where endotoxins are manifestly present during the abnormal or pathological disease state in a living subject. Endotoxin is usually present in large quantities in the gastro-intestinal tract in the normal condition. No harmful effects are produced; and endotoxin is either not detectable or is present in extremely low concentrations (&lt;10 pg/ml) in the circulating plasma or serum of healthy individuals. Even the intentional ingestion and introduction of milligram quantities of endotoxin fails to produce adverse reactions in healthy subjects [Bayston, K. F. and J. Cohen, J. Med, Microbiol. 31:73-83 (1990)]. This is believed to be due largely to the fact that the gut mucosa of healthy living individuals is both impervious to and resistant to the effects of intestinal endotoxins.
In general, however, there are three routes by which endotoxins (LPS) enter the tissues and organs of the body. These are: via the portal vein; by direct transmural absorption into the systemic blood stream; or by the intestinal lymphatics. Of these, the first two routes are considered the most important in man. Thus, if the gastrointestinal mucosa, and portal circulation, and hepatic-reticuloendotheoil system are intact, endotoxins do not appear in the blood stream and do not cause damage to organs and tissues. In contrast, the reported empirical evidence has revealed that if the gut is damaged or made permeable by any number of different events or disease states, endotoxins will transmigrate through the bowel mucosa [Aoki, K. A., Acta. Med. Okayama 32:207-216 (1978); Papa et al., J. Surg. Res. 35:264-269 (1983); Walker, R. I., Exp. Hematol. 6:172-184 (1978)]. Thus, if the gut is damaged or there is increased mucosal permeability of endotoxin secondary to systemic disease, endotoxin levels in blood will increase and give rise to the systemic effects associated with endotoxemia. Accordingly, symptoms typical of endotoxemia such as fever, hypotension, respiratory distress, hypercoagulability, and cholestasis, which complicate many inflammatory and ischemic diseases of the bowel as well as other disorders, are thought to be due to enhanced resorption of endotoxins [Van DeVenter et al., Gastroenterology 94:825-831 (1988)]. There is also considerable evidence that the transmigration of endotoxin through the gut is a commonly shared event which occurs in many different disease states. These include: bowel ischemia; peritonitis; total body irradiation consequences; colitis; portal vein occlusion; various forms of acute and chronic liver diseases; Crohn's disease; ulcerative colitis; necrotizing enterocolitis; and systemic infections of children and adults.
A number of widely used methods of varying sensitivity for detecting endotoxin are known. Perhaps the most widely used and most sensitive method is the Limulus amoebocyte lysate (LAL) assay [Jorgenson, J. H., Handbook of Endotoxin (Proctor, R. A., editor), volume 4, Elsevier, Amsterdam, 1986, page 1270; Yokota et al., J. Biochem. Biophys. Meth. 18:97-104 (1989)]. The principle of this assay is that gelation occurs when a sample containing endotoxin causes activation of a series of primitive enzymes present in the lysate of the horseshoe crab, Limulus polythemus. All the factors necessary for activation of the clotting process are found within the granules called amoebocytes; and with a lysate of the cells, a simple, gel-clot test able to detect picogram quantities of endotoxin is available.
In addition to the gel-clot method, several other variant assay methods for the detection of endotoxin have been introduced. These are based upon endotoxin-induced LAL activation; or upon turbidimetric and nephelometric measurements of the gelation reaction; or a determination of the protein content of the gel clot; or upon rocket immunoelectrophoresis; or on a direct measurement of the action of activated clotting enzyme on a synthetic chromogenic substrate.
The recognized high incidence of endotoxin mediated injury in vivo, and the all-too frequent mortality associated therewith, have led to investigations of therapeutic treatments and options. Among the reported investigations and attempts to neutralize the effects of endotoxin, three general forms of therapy for counteracting the effects of endotoxin have been pursued. These are: neutralizing the effect of endotoxin on macrophage mediators; decreasing intestinal production and absorption of endotoxin; and using monoclonal antibodies specific for endotoxin. Details for each of these three individual strategies and approaches are presented by Table B below.